Folate deficiency is a common cause of megaloblastic anemia in man. In the human body, folic acid is metabolized to tetrahydrofolic acid and subsequently to N-5-methyltetrahydrofolic acid, which in turn can be reconverted to tetrahydrofolic acid in the presence of vitamin B.sub.12 to reenter the metabolic pool of 1-carbon fragment donors and acceptors. It is important to be able to measure the amount of N-5-methyltetrahydrofolic acid in the serum, plasma and/or red blood cells.
Quite often, the concentration of N-5-methyltetrahydrofolic acid, the major folate derivative in blood, is measured using a competitive protein binding assay. As will be explained more fully hereinbelow, a competitive protein binding assay for N-5-methyltetrahydrofolic acid should preferably include the utilization of N-5-methyltetrahydrofolic acid in the standard reagents. However, N-5-methyltetrahydrofolic acid is very unstable, requiring measures such as sealing in ampoules in lyophilized form under a nitrogen atmosphere for proper storage. Such techniques are very impractical in the manufacture of an assay reagent kit. Furthermore, hermetic sealing could not prolong stability after reconstitution, which for N-5-methyltetrahydrofolic acid is only about three days at 4.degree. C.
Because of the relationship between vitamin B.sub.12 and folid acid in man, assays have been developed which are used to determine not only the N-5-methyltetrahydrofolic acid level, but also the concentration of vitamin B.sub.12 using a single multi-step assay procedure. See the protocol of U.S. Pat. No. 4,146,602 for a purported description of a simultaneous vitamin B.sub.12 /folate radioassay. In fact, since vitamin B.sub.12 and folate deficiencies are hematologically and clinically indistinguishable, it is usually necessary to determine the level of vitamin B.sub.12 in the serum and the level of folate in the serum and red blood cells to help establish definitively the etiology of megaloblastic anemia.
Among various preservative compounds which can be employed to stabilize N-5-methyltetrahydrofolic acid, ascorbic acid is preferred. However, ascorbic acid is known to interfere with competitive binding vitamin B.sub.12 analysis. Because of the known instability of N-5-methyltetrahydrofolic acid, many folate assays requiring folate standard(s), particularly using a simultaneous vitamin B.sub.12 /folate assay technique, have used the parent compound, folic acid, as the folate standard. However, folic acid does not always duplicate the activity of N-5-methyltetrahydrofolic acid in the testing procedure.